基于介孔碳材料的电化学生物传感器研究
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摘要
生物传感器作为一门涉及化学、生物学、物理学以及电子学等领域的交叉学科,在临床医学、工农业生产和环境保护等诸多领域有着广阔的应用前景。生物传感技术也必将是21世纪知识经济发展中介于信息和生物技术之间的新增长点。在生物传感器的发展进程中,电化学生物传感器是十分重要的一类。它是由生物材料作为敏感元件,电极作为转换元件,以电势、电流或电导等作为特征检测信号的传感器。其研制过程中的一个关键因素是生物分子的固定化。如何在电极表面有效地固定生物分子,无论是对于研究蛋白质等生物分子的性质,还是研制新型电化学生物传感器都至关重要。理想的生物分子的固定方法要求既能促进有效的电子转移,又能保持被固定生物分子的活性。
碳基纳米材料以其良好的导电性和化学稳定性吸引了越来越多的关注并得到了广泛的研究。然而在典型的氧化还原蛋白质/电极的生物电化学研究中,氧化还原点位同电极表面之间需要通过介体来促进电子转移。当没有介体存在时,仅有少数氧化还原蛋白质在碳纳米管修饰的电极上表现出直接电子转移。因此研究氧化还原蛋白质的直接电子转移对于蛋白质氧化还原性质和生物传感器的基础研究都有重要意义。
众所周知主要有两个因素影响固定化蛋白质的氧化还原行为和生物活性。一是载体对蛋白质的负载能力;一是载体材料的生物相容性和电催化性能。纳米材料如纳米粒子、碳纳米管、纳米孔金属氧化物等,正由于能够有效固定生物组分,在生物传感器中具有极大的应用潜力,日益引起人们的关注。近年来随着介孔材料的兴起和发展,人们合成出介孔碳材料(CMMs),这种新型的碳纳米材料在催化剂载体、吸附剂及电子器件等方面被广泛应用。CMMs是由具有高度有序排列以及大孔隙率的碳纳米棒所组成,不仅像碳纳米管一样具有良好的电学性质和化学稳定性,也表现出很多独特性质,例如高度有序的孔结构、易于调控的介观结构、狭小的孔径分布、更大的比表面积(高达2000 m~2 g~(-1))和比孔容(可达1.5 cm~3 g~(-1))等。而且还可以通过调控介孔碳材料的孔道尺寸、介观拓扑结构和表面荷电情况来设计适应不同生物分子固定化需求的载体。这都使介孔碳材料在蛋白质固定和生物传感器研究等方面拥有极大的优势和应用前景。此外,CMMs可以很容易的通过硬模板法制备,成本低廉,同时在二氧化硅“模板”被完全刻蚀的情况下无任何杂质污染。然而到目前为止,CMMs很多其他的潜在优势,如生物相容性和电催化性质尤其是在固载蛋白质和制备生物传感器方面的应用报道较少。
通过将新型纳米材料修饰到电极表面,可以有效地固定生物分子,并促进其氧化还原中心与电极之间的直接电子转移,从而研制新一代的生物传感器及其他生物器件。因此,本课题具有较为重大的理论研究和实际应用意义。
本论文共分为六个部分:
第一章,首先对纳米材料和生物传感器的研究现状和发展前景进行了简述。接着在此基础上提出本论文的研究设想,即采用功能纳米材料修饰的电极来固定蛋白质分子,通过电化学方法来研究蛋白质的电子转移、电催化和生物传感应用。着重介绍了基于介孔碳及其纳米掺杂复合物的蛋白质固定化技术。最后,提出了本论文的实验思路和研究意义。
第二章,研究了基于铂纳米粒子掺杂介孔碳的蛋白质电化学和生物传感应用。铂纳米粒子(Platinum nanoparticles,PtNPs)具有良好的导电性和催化性质,可以有效的提高所制备的纳米复合物的导电性和电催化活性。与未掺杂的二维介孔碳(2D-CMM)相比,分散了铂纳米粒子的介孔碳修饰电极由于两种材料促进电子传递和提高电催化性能方面的协同效应,能促进其固定化酶的氧化还原可逆性。在此以葡萄糖氧化酶为模型,制备了铂纳米粒子掺杂的介孔碳纳米复合物薄膜(Pt-CMM),用来研究固定化酶的准可逆的电子传递,其表观的异相电子传递速率(k_(et)~0)为6.5 s~(-1),高于纯介孔碳固定化酶的3.9 s~(-1)。同时,进一步考察了该传感器的生物催化活性,当铂/介孔碳质量百分比为5%时,传感器具有较好的稳定性,对葡萄糖响应迅速灵敏,且具有更低的检测下限。由于贵金属纳米颗粒掺杂所带来的成本的升高和稀缺资源的消耗,我们接下来将着重从介孔碳的结构调控等方面进行深入的研究。
第三章,首先考察了基于二维和三维有序介孔碳的蛋白质电化学和生物传感器研究。介孔碳复合物基质既能较好的固定酶分子,又能为固定化的酶分子提供生物相容的微环境以保持其生物活性。同时,由于其固有的高导电性,介孔碳材料有助于促进固定化酶分子与基底电极之间的电子传递。由此设计并制备了二维和三维高度有序的介孔碳用于固定葡萄糖氧化酶分子以研究其准可逆的电子传递,实验测得其异相电子传递速率值分别为3.9和4.2 s~(-1)。此外还进一步研究了传感器的生物催化活性。与二维有序的介孔碳材料相比,三维有序的介孔碳材料对蛋白质表现出更高的负载能力,其固定化的酶分子保持了更高的生物活性,制备的葡萄糖生物传感器具有灵敏度高、线性范围宽和检测限低等特点。
第四章,研究了基于双连续螺旋介孔碳的蛋白质电化学和生物传感器研究。由于二维有序的介孔碳存在某一个空间维度的无序性,导致了其导电的各向异性比三维有序介孔碳大。故上一章中三维介孔碳比二维介孔碳的表观导电性高,然而这种提高程度还不是非常理想。这是由于合成三维介孔碳的某些氧化硅模板如MCM-48、FDU-5等缺少相互连接的微孔,当模板被刻蚀除去以后会发生碳骨架结构的部分错位,导致空间对称性的下降同时其导电各向异性程度增强,从而使上述碳基材料对蛋白质与电极之间电子转移的促进作用在一定程度上受到限制。而双连续螺旋介孔碳(Bicontinuous gyroidal mesoporous carbon,BGMC)则是一种具有更高有序度更高空间对称性——立方Ia(?)d结构的三维介孔碳,从而具有相对各向同性的石墨化结构和电导能力,因此可以更为有效的促进异相电子转移。为此设计构建了BGMC纳米复合物薄膜固定蛋白质(以葡萄糖氧化酶和肌红蛋白为模型),研究了蛋白质准可逆的电子传递并揭示了其较高的生物催化活性。还进一步设计制备了一系列不同孔径(2-7 nm)的以蔗糖或酚醛树脂(Phenolformaldehyde,PF)作为碳源的BGMC,研究其孔径和碳源对氧化还原蛋白质固定和异相电子传递的影响。实验结果表明BGMC的不同孔径和碳源对蛋白质固定及其电化学性质具有较大影响。这也意味着可以通过调控介孔碳的孔径、拓扑结构等来制备适宜于不同尺度生物分子的固定基质,以促进异相电子转移并提高固定化酶的生物电催化性能。这些性质使BGMC在研究蛋白质直接电化学方面极具价值,同时拓宽了碳基生物传感器的发展途径。
第五章,研究了基于双连续螺旋介孔碳的NADH的电化学氧化。到目前为止,关于利用三维介孔碳材料进行NADH直接电化学氧化的研究尚无报道。于是本章利用BGMC修饰的玻碳电极来研究NADH的电化学氧化。BGMC所具有的大比表面积和高电子传递能力以及丰富的边片状缺陷位和表面的含氧官能团,对NADH的氧化表现出了较高的电催化性能,将NADH的氧化过电位降低了649mV(与未修饰的裸玻碳电极相比)。BGMC修饰电极实现了NADH在低电位条件下(+0.046 V vs.SCE,pH 7.2)稳定灵敏的响应。检测下限为1.0×10~(-6) M,响应范围较宽(3.0×10~(-6)-1.4×10~(-3) M)。这为基于以NADH为电活性物质的脱氢酶的电流型生物传感器的发展提供了新的研究途径。
第六章,对全文进行了总结,指出了研究中存在的一些不足,并提出了后续工作的目标和研究思路。
Biosensors,as an interdisciplinary frontier related to chemistry,biology,physics, medical science and electronics,will have broad applications in clinical medicine, industry,agriculture and environment protection.Biosensing technology will also become the new growing point between information technology and biological technology during the development of intelligent economy in 21 century. Electrochemical biosensor is extensively investigated and becomes one of the most practical and prospective devices among all kinds of biosensors.It is composed of the biomaterial as a receptor and an electrode as a transducer,detecting the generated electrical signals.A key factor in biosensor development is the immobilization of biomolecules.It is very significant to find a reliable strategy to immobilize biomolecules,retaining their high activity and meanwhile allowing the effective electron-transfer(ET) between the redox centers and the electrode surface.
Carbon based nanomaterials have drawn increasing attention because of their good conductivity and chemical stability.However,bioelectrochemical studies of redox proteins/electrode typically require mediators to promote ET between the redox sites and the electrode surface.Hence,when no mediators present,only a few redox proteins performed direct ET on electrodes modified with carbon nanotubes(CNTs). Thus it is important for the understanding on protein properties and the development of bioelectrochemical devices to study the direct ET of redox proteins.
It is well known that two factors seriously affect the redox behavior and bio-activity of the immobilized proteins.One is the protein-loading capability of the material;the other is the biocompatibility and the electro-catalytic performance of the matrix.Nanomaterials,such as nanoparticles,CNTs,nanoporous metal oxides and so on,can immobilize bio-components effectively and have great potentials in the application of biosensors,arousing increasing research interest.Carbon mesoporous materials(CMMs),a new type of nanostructured carbon materials,have recently shown many unique properties and attracted growing interest in diverse fields from catalyst carriers,absorbents to electronic devices.CMMs are composed of carbon nanorods with highly ordered arrays.Thus CMMs not only have good conductivity and chemical stability like CNTs,but also show many unique properties,such as highly-ordered and tailored mesostructures,much narrow pore size distributions,large surface areas(up to ca.2000 m~2 g~(-1)) and large porosity(up to ca.1.5 cm~3 g~(-1)).That means it is possible to design the selectivity of substrates for biomolecule immobilization,by varying the pore diameter,mesoscopic topology and charge of CMMs.All of these advantages make them greatly promising in the realm of protein immobilization and heterogeneous ET.Besides,CMMs can be easily prepared via the hard templating strategy with low cost and free from any impurities if silica 'mold' is fully etched away.While up to now,many other potential advantages of CMMs,e.g. biocompatibility and eletrocatalytic properties,have rarely been addressed,especially for its applications in immobilizing proteins and fabricating biosensors.
The electrode modification with novel nanomaterials can immobilize biomolecules effectively and promote the direct ET between redox centers and electrodes,thus develop a new generation of biosensors and other bio-devices. Therefore,this issue is of great theoretical and practical significance.
This dissertation includes six chapters as follows:
In Chapter One,we introduced the research and development of nanomaterials and biosensors,respectively.Then we proposed a scheme of functional nanomaterials-modified electrodes for protein immobilization and research on ET, electro-catalysis and biosensing.Finally we outlined the experimental ideas and the research purposes of this thesis.
In Chapter Two,we studied the electrochemistry and biosensing of glucose oxidase(GOx) based on Pt-dispersed CMM.Platinum nanoparticles(PtNPs) provided with excellent conductivity and catalytic properties can effectively enhance conductive and electrocatalytic performance of the prepared nanocomposite. Compared with the pure two dimensional(2D-) CMM,CMM dispersed with PtNPs enhances the electron communication and redox reversibility of redox enzyme at the modified electrode,due to the cooperative effect of PtNPs and CMM on the ET promotion and electrocatalytic activity.Based on Pt-CMM nanocomposite film,the quasi-reversible electron transfer for GOx is probed and the apparent heterogeneous electron transfer rate constant(k_(et)~0) is 6.5 s~(-1),lager than 3.9 s~(-1) based on pure CMM. In addition,the associated biocatalytic activity is revealed.The prepared biosensor offers a more stable and sensitive amperometric detection of glucose with a lower detection limit under the optimal Pt/CMM weight ratio of 5 wt.%.Considering the high cost and resource consumption when doping noble metal nanopaticles,we focused on the deep study in the mesostructure adjustment in the following work.
In Chapter Three,we investigate the electrochemistry and biosensing of GOx based on CMMs with different spatially-ordered dimensions.As discussed in Chapter Two,the CMM composite matrix is provided with both the ideal immobilization ability for enzymes and the biocompatible microenvironment for preserving the bioactivity of enzyme molecules.Meanwhile,due to their inherent good conductivity, the CMMs can promote the electron communication between the immobilized enzyme molecules and the underlying electrode,realizing fast electron transfer between the enzymes and the modified electrode surface.Thus we designed and prepared highly ordered two-dimensional(2D-) and three-dimensional(3D-) CMMs to immobilize GOx.The quasi-reversible ET of the redox enzyme is probed,and the apparent heterogeneous electron transfer rate constants(k_(et)~0) are 3.9 and 4.2 s~(-1) respectively.Furthermore,the associated biocatalytic activity was also revealed. Highly ordered 3D-CMM exhibited larger adsorption capacity for proteins and the immobilized enzymes retained a higher bioactivity compared with 2D-CMM.The mediated glucose biosensor based on 3D-CMM showed a high sensitivity,broad linear response range,and low detection limit.
In Chapter Four,we studied the electrochemistry and biosensing of proteins based on bicontinuous gyroidal mesoporous carbon(BGMC).Because 2D-CMM has a disordered dimension,resulting in its conductivity anisotropy higher than that of 3D-CMM,3D-CMM exhibits a higher apparent conductivity than 2D-CMM. However,such enhancing degree is not very ideal.Because some silica templates of 3D-CMM,such as MCM-48 and FDU-5,are lack of interconnected micropores,the partial displacement of the carbon frameworks occurs after the removal of the silica template,leading to the decrease in spatial symmetry and simultaneously the increase in the conductivity amsotropy.Thus the above carbon matrixes are limited in achieving a considerably ideal promotion of ET between redox proteins and electrodes.While BGMC has a more ordered mesoscopic structure with the higher symmetry of 3D-Ia(?)d,possessing a relatively isotropic graphited structure and thus can more effectively enhance the heterogeneous electron communication.Herein a strategy was demonstrated,of the preparation of protein-entrapped BGMC nanocomposite films for assembling redox proteins(employed glucose oxidase and myoglobin as the models).The immobilized proteins showed fast electrochemistry and retained their bioactivity to a certain extent.In addition,a series of BGMCs with different pore sizes from 2 to 7 nm was designed and synthesized with sucrose or phenol formaldehyde(PF) resin as a carbon source.Pore sizes and carbon sources show great influences on the immobilization of redox proteins and on the ET between redox proteins and electrodes.It means that the pore diameter and structural topologies can be readily tuned to match the dimensional size of diverse biomolecules, to promote heterogeneous ET and to enhance the electrocatalytic properties of the proteins.All these properties make BGMC valuable in the understanding on the electrochemistry of redox proteins and expand the scope of carbon-based electrochemical devices.
In Chapter Five,we studied the electrocatalytic oxidation of NADH based on BGMC with low overpotential.To the best of our knowledge,no previous work has been reported on the direct electro-oxidation research of NADH based on 3D-CMM. Herein we used the BGMC-modified electrode to study the electrochemical oxidation of NADH.The large specific surface area and the high electron-communicating capability of BGMC,plus the considerable edge-plane-like defective sites and the oxygen-containing groups on the BGMC surface could be responsible for its electrocatalytic behavior,which induced a substantial decrease by 649 mV in the overpotential of NADH oxidation reaction(compared with a bare electrode).The BGMC-modified electrode offers a stable and sensitive amperometric detection of NADH at a low overpotential(+0.046 V vs.SCE,pH 7.2) with a low detection limit (1.0×10~(-6) M) and a broad linear response range(3.0×10~(-6)～1.4×10~(-3) M).This might broaden the development avenue of amperometric biosensors based on NADH-dependent dehydrogenase.
In Chapter Six,we summarized and pointed out the shortcomings in this dissertation.Finally we proposed the objects and schemes of further research.
碳基纳米材料以其良好的导电性和化学稳定性吸引了越来越多的关注并得到了广泛的研究。然而在典型的氧化还原蛋白质/电极的生物电化学研究中,氧化还原点位同电极表面之间需要通过介体来促进电子转移。当没有介体存在时,仅有少数氧化还原蛋白质在碳纳米管修饰的电极上表现出直接电子转移。因此研究氧化还原蛋白质的直接电子转移对于蛋白质氧化还原性质和生物传感器的基础研究都有重要意义。
众所周知主要有两个因素影响固定化蛋白质的氧化还原行为和生物活性。一是载体对蛋白质的负载能力;一是载体材料的生物相容性和电催化性能。纳米材料如纳米粒子、碳纳米管、纳米孔金属氧化物等,正由于能够有效固定生物组分,在生物传感器中具有极大的应用潜力,日益引起人们的关注。近年来随着介孔材料的兴起和发展,人们合成出介孔碳材料(CMMs),这种新型的碳纳米材料在催化剂载体、吸附剂及电子器件等方面被广泛应用。CMMs是由具有高度有序排列以及大孔隙率的碳纳米棒所组成,不仅像碳纳米管一样具有良好的电学性质和化学稳定性,也表现出很多独特性质,例如高度有序的孔结构、易于调控的介观结构、狭小的孔径分布、更大的比表面积(高达2000 m~2 g~(-1))和比孔容(可达1.5 cm~3 g~(-1))等。而且还可以通过调控介孔碳材料的孔道尺寸、介观拓扑结构和表面荷电情况来设计适应不同生物分子固定化需求的载体。这都使介孔碳材料在蛋白质固定和生物传感器研究等方面拥有极大的优势和应用前景。此外,CMMs可以很容易的通过硬模板法制备,成本低廉,同时在二氧化硅“模板”被完全刻蚀的情况下无任何杂质污染。然而到目前为止,CMMs很多其他的潜在优势,如生物相容性和电催化性质尤其是在固载蛋白质和制备生物传感器方面的应用报道较少。
通过将新型纳米材料修饰到电极表面,可以有效地固定生物分子,并促进其氧化还原中心与电极之间的直接电子转移,从而研制新一代的生物传感器及其他生物器件。因此,本课题具有较为重大的理论研究和实际应用意义。
本论文共分为六个部分:
第一章,首先对纳米材料和生物传感器的研究现状和发展前景进行了简述。接着在此基础上提出本论文的研究设想,即采用功能纳米材料修饰的电极来固定蛋白质分子,通过电化学方法来研究蛋白质的电子转移、电催化和生物传感应用。着重介绍了基于介孔碳及其纳米掺杂复合物的蛋白质固定化技术。最后,提出了本论文的实验思路和研究意义。
第二章,研究了基于铂纳米粒子掺杂介孔碳的蛋白质电化学和生物传感应用。铂纳米粒子(Platinum nanoparticles,PtNPs)具有良好的导电性和催化性质,可以有效的提高所制备的纳米复合物的导电性和电催化活性。与未掺杂的二维介孔碳(2D-CMM)相比,分散了铂纳米粒子的介孔碳修饰电极由于两种材料促进电子传递和提高电催化性能方面的协同效应,能促进其固定化酶的氧化还原可逆性。在此以葡萄糖氧化酶为模型,制备了铂纳米粒子掺杂的介孔碳纳米复合物薄膜(Pt-CMM),用来研究固定化酶的准可逆的电子传递,其表观的异相电子传递速率(k_(et)~0)为6.5 s~(-1),高于纯介孔碳固定化酶的3.9 s~(-1)。同时,进一步考察了该传感器的生物催化活性,当铂/介孔碳质量百分比为5%时,传感器具有较好的稳定性,对葡萄糖响应迅速灵敏,且具有更低的检测下限。由于贵金属纳米颗粒掺杂所带来的成本的升高和稀缺资源的消耗,我们接下来将着重从介孔碳的结构调控等方面进行深入的研究。
第三章,首先考察了基于二维和三维有序介孔碳的蛋白质电化学和生物传感器研究。介孔碳复合物基质既能较好的固定酶分子,又能为固定化的酶分子提供生物相容的微环境以保持其生物活性。同时,由于其固有的高导电性,介孔碳材料有助于促进固定化酶分子与基底电极之间的电子传递。由此设计并制备了二维和三维高度有序的介孔碳用于固定葡萄糖氧化酶分子以研究其准可逆的电子传递,实验测得其异相电子传递速率值分别为3.9和4.2 s~(-1)。此外还进一步研究了传感器的生物催化活性。与二维有序的介孔碳材料相比,三维有序的介孔碳材料对蛋白质表现出更高的负载能力,其固定化的酶分子保持了更高的生物活性,制备的葡萄糖生物传感器具有灵敏度高、线性范围宽和检测限低等特点。
第四章,研究了基于双连续螺旋介孔碳的蛋白质电化学和生物传感器研究。由于二维有序的介孔碳存在某一个空间维度的无序性,导致了其导电的各向异性比三维有序介孔碳大。故上一章中三维介孔碳比二维介孔碳的表观导电性高,然而这种提高程度还不是非常理想。这是由于合成三维介孔碳的某些氧化硅模板如MCM-48、FDU-5等缺少相互连接的微孔,当模板被刻蚀除去以后会发生碳骨架结构的部分错位,导致空间对称性的下降同时其导电各向异性程度增强,从而使上述碳基材料对蛋白质与电极之间电子转移的促进作用在一定程度上受到限制。而双连续螺旋介孔碳(Bicontinuous gyroidal mesoporous carbon,BGMC)则是一种具有更高有序度更高空间对称性——立方Ia(?)d结构的三维介孔碳,从而具有相对各向同性的石墨化结构和电导能力,因此可以更为有效的促进异相电子转移。为此设计构建了BGMC纳米复合物薄膜固定蛋白质(以葡萄糖氧化酶和肌红蛋白为模型),研究了蛋白质准可逆的电子传递并揭示了其较高的生物催化活性。还进一步设计制备了一系列不同孔径(2-7 nm)的以蔗糖或酚醛树脂(Phenolformaldehyde,PF)作为碳源的BGMC,研究其孔径和碳源对氧化还原蛋白质固定和异相电子传递的影响。实验结果表明BGMC的不同孔径和碳源对蛋白质固定及其电化学性质具有较大影响。这也意味着可以通过调控介孔碳的孔径、拓扑结构等来制备适宜于不同尺度生物分子的固定基质,以促进异相电子转移并提高固定化酶的生物电催化性能。这些性质使BGMC在研究蛋白质直接电化学方面极具价值,同时拓宽了碳基生物传感器的发展途径。
第五章,研究了基于双连续螺旋介孔碳的NADH的电化学氧化。到目前为止,关于利用三维介孔碳材料进行NADH直接电化学氧化的研究尚无报道。于是本章利用BGMC修饰的玻碳电极来研究NADH的电化学氧化。BGMC所具有的大比表面积和高电子传递能力以及丰富的边片状缺陷位和表面的含氧官能团,对NADH的氧化表现出了较高的电催化性能,将NADH的氧化过电位降低了649mV(与未修饰的裸玻碳电极相比)。BGMC修饰电极实现了NADH在低电位条件下(+0.046 V vs.SCE,pH 7.2)稳定灵敏的响应。检测下限为1.0×10~(-6) M,响应范围较宽(3.0×10~(-6)-1.4×10~(-3) M)。这为基于以NADH为电活性物质的脱氢酶的电流型生物传感器的发展提供了新的研究途径。
第六章,对全文进行了总结,指出了研究中存在的一些不足,并提出了后续工作的目标和研究思路。
Biosensors,as an interdisciplinary frontier related to chemistry,biology,physics, medical science and electronics,will have broad applications in clinical medicine, industry,agriculture and environment protection.Biosensing technology will also become the new growing point between information technology and biological technology during the development of intelligent economy in 21 century. Electrochemical biosensor is extensively investigated and becomes one of the most practical and prospective devices among all kinds of biosensors.It is composed of the biomaterial as a receptor and an electrode as a transducer,detecting the generated electrical signals.A key factor in biosensor development is the immobilization of biomolecules.It is very significant to find a reliable strategy to immobilize biomolecules,retaining their high activity and meanwhile allowing the effective electron-transfer(ET) between the redox centers and the electrode surface.
Carbon based nanomaterials have drawn increasing attention because of their good conductivity and chemical stability.However,bioelectrochemical studies of redox proteins/electrode typically require mediators to promote ET between the redox sites and the electrode surface.Hence,when no mediators present,only a few redox proteins performed direct ET on electrodes modified with carbon nanotubes(CNTs). Thus it is important for the understanding on protein properties and the development of bioelectrochemical devices to study the direct ET of redox proteins.
It is well known that two factors seriously affect the redox behavior and bio-activity of the immobilized proteins.One is the protein-loading capability of the material;the other is the biocompatibility and the electro-catalytic performance of the matrix.Nanomaterials,such as nanoparticles,CNTs,nanoporous metal oxides and so on,can immobilize bio-components effectively and have great potentials in the application of biosensors,arousing increasing research interest.Carbon mesoporous materials(CMMs),a new type of nanostructured carbon materials,have recently shown many unique properties and attracted growing interest in diverse fields from catalyst carriers,absorbents to electronic devices.CMMs are composed of carbon nanorods with highly ordered arrays.Thus CMMs not only have good conductivity and chemical stability like CNTs,but also show many unique properties,such as highly-ordered and tailored mesostructures,much narrow pore size distributions,large surface areas(up to ca.2000 m~2 g~(-1)) and large porosity(up to ca.1.5 cm~3 g~(-1)).That means it is possible to design the selectivity of substrates for biomolecule immobilization,by varying the pore diameter,mesoscopic topology and charge of CMMs.All of these advantages make them greatly promising in the realm of protein immobilization and heterogeneous ET.Besides,CMMs can be easily prepared via the hard templating strategy with low cost and free from any impurities if silica 'mold' is fully etched away.While up to now,many other potential advantages of CMMs,e.g. biocompatibility and eletrocatalytic properties,have rarely been addressed,especially for its applications in immobilizing proteins and fabricating biosensors.
The electrode modification with novel nanomaterials can immobilize biomolecules effectively and promote the direct ET between redox centers and electrodes,thus develop a new generation of biosensors and other bio-devices. Therefore,this issue is of great theoretical and practical significance.
This dissertation includes six chapters as follows:
In Chapter One,we introduced the research and development of nanomaterials and biosensors,respectively.Then we proposed a scheme of functional nanomaterials-modified electrodes for protein immobilization and research on ET, electro-catalysis and biosensing.Finally we outlined the experimental ideas and the research purposes of this thesis.
In Chapter Two,we studied the electrochemistry and biosensing of glucose oxidase(GOx) based on Pt-dispersed CMM.Platinum nanoparticles(PtNPs) provided with excellent conductivity and catalytic properties can effectively enhance conductive and electrocatalytic performance of the prepared nanocomposite. Compared with the pure two dimensional(2D-) CMM,CMM dispersed with PtNPs enhances the electron communication and redox reversibility of redox enzyme at the modified electrode,due to the cooperative effect of PtNPs and CMM on the ET promotion and electrocatalytic activity.Based on Pt-CMM nanocomposite film,the quasi-reversible electron transfer for GOx is probed and the apparent heterogeneous electron transfer rate constant(k_(et)~0) is 6.5 s~(-1),lager than 3.9 s~(-1) based on pure CMM. In addition,the associated biocatalytic activity is revealed.The prepared biosensor offers a more stable and sensitive amperometric detection of glucose with a lower detection limit under the optimal Pt/CMM weight ratio of 5 wt.%.Considering the high cost and resource consumption when doping noble metal nanopaticles,we focused on the deep study in the mesostructure adjustment in the following work.
In Chapter Three,we investigate the electrochemistry and biosensing of GOx based on CMMs with different spatially-ordered dimensions.As discussed in Chapter Two,the CMM composite matrix is provided with both the ideal immobilization ability for enzymes and the biocompatible microenvironment for preserving the bioactivity of enzyme molecules.Meanwhile,due to their inherent good conductivity, the CMMs can promote the electron communication between the immobilized enzyme molecules and the underlying electrode,realizing fast electron transfer between the enzymes and the modified electrode surface.Thus we designed and prepared highly ordered two-dimensional(2D-) and three-dimensional(3D-) CMMs to immobilize GOx.The quasi-reversible ET of the redox enzyme is probed,and the apparent heterogeneous electron transfer rate constants(k_(et)~0) are 3.9 and 4.2 s~(-1) respectively.Furthermore,the associated biocatalytic activity was also revealed. Highly ordered 3D-CMM exhibited larger adsorption capacity for proteins and the immobilized enzymes retained a higher bioactivity compared with 2D-CMM.The mediated glucose biosensor based on 3D-CMM showed a high sensitivity,broad linear response range,and low detection limit.
In Chapter Four,we studied the electrochemistry and biosensing of proteins based on bicontinuous gyroidal mesoporous carbon(BGMC).Because 2D-CMM has a disordered dimension,resulting in its conductivity anisotropy higher than that of 3D-CMM,3D-CMM exhibits a higher apparent conductivity than 2D-CMM. However,such enhancing degree is not very ideal.Because some silica templates of 3D-CMM,such as MCM-48 and FDU-5,are lack of interconnected micropores,the partial displacement of the carbon frameworks occurs after the removal of the silica template,leading to the decrease in spatial symmetry and simultaneously the increase in the conductivity amsotropy.Thus the above carbon matrixes are limited in achieving a considerably ideal promotion of ET between redox proteins and electrodes.While BGMC has a more ordered mesoscopic structure with the higher symmetry of 3D-Ia(?)d,possessing a relatively isotropic graphited structure and thus can more effectively enhance the heterogeneous electron communication.Herein a strategy was demonstrated,of the preparation of protein-entrapped BGMC nanocomposite films for assembling redox proteins(employed glucose oxidase and myoglobin as the models).The immobilized proteins showed fast electrochemistry and retained their bioactivity to a certain extent.In addition,a series of BGMCs with different pore sizes from 2 to 7 nm was designed and synthesized with sucrose or phenol formaldehyde(PF) resin as a carbon source.Pore sizes and carbon sources show great influences on the immobilization of redox proteins and on the ET between redox proteins and electrodes.It means that the pore diameter and structural topologies can be readily tuned to match the dimensional size of diverse biomolecules, to promote heterogeneous ET and to enhance the electrocatalytic properties of the proteins.All these properties make BGMC valuable in the understanding on the electrochemistry of redox proteins and expand the scope of carbon-based electrochemical devices.
In Chapter Five,we studied the electrocatalytic oxidation of NADH based on BGMC with low overpotential.To the best of our knowledge,no previous work has been reported on the direct electro-oxidation research of NADH based on 3D-CMM. Herein we used the BGMC-modified electrode to study the electrochemical oxidation of NADH.The large specific surface area and the high electron-communicating capability of BGMC,plus the considerable edge-plane-like defective sites and the oxygen-containing groups on the BGMC surface could be responsible for its electrocatalytic behavior,which induced a substantial decrease by 649 mV in the overpotential of NADH oxidation reaction(compared with a bare electrode).The BGMC-modified electrode offers a stable and sensitive amperometric detection of NADH at a low overpotential(+0.046 V vs.SCE,pH 7.2) with a low detection limit (1.0×10~(-6) M) and a broad linear response range(3.0×10~(-6)～1.4×10~(-3) M).This might broaden the development avenue of amperometric biosensors based on NADH-dependent dehydrogenase.
In Chapter Six,we summarized and pointed out the shortcomings in this dissertation.Finally we proposed the objects and schemes of further research.
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